Topoisomerase II (Top2) is the target of a wide range of active anti-cancer agents. Drugs targeting Top2 are widely used and active agents, but a detailed understanding of how they act against the enzyme has proven elusive. Clinically active Top2 targeting agents lead to enzyme mediated DNA damage, and are referred to as Top2 poisons. Detailed information about the action of Top2 poisons has the potential for leading to safer and more active anti-cancer drugs. An important model system for understanding the biological and biochemical properties of DNA topoisomerases is the yeast Saccharomyces cerevisiae. The genetic tools available in this organism also make it a powerful system for studying mechanisms of action of anti-cancer drugs targeting topoisomerases. Yeast Top2 has also been the enzyme of choice for structural studies of eukaryotic Top2. The overall goal of this project is to apply all of the tools available with the yeast system to obtain a detailed understanding of how Top2 poisons interact with the enzyme, and how Top2 drug interactions lead to Top2 mediated DNA damage. We propose a combination of genetic, biochemical and structural approaches to understand the action of Top2 poisons on their target enzymes. Genetic studies will allow the isolation and characterization of novel drug hypersensitive mutants of Top2. These studies will increase our understanding of how Top2 poisons bind to the enzyme and generate enzyme-mediated DNA damage. Assays for assessing drug action, including measurements of drug affinity and enzyme mediated DNA cleavage will be applied to test how drug hypersensitivity occurs in the mutant proteins. We also plan to study a novel mutant of human Top2 that mimics the action of Top2 poisons in the absence of drug. Studies with this mutant will provide a biochemical understanding of Top2 mediated DNA damage in the context of the overall reaction cycle. Finally, the drug hypersensitive mutants will provide necessary reagents for structural studies that will attempt to solve the structure of drug:protein:DNA ternary complexes. The proposed experiments will be useful in the design of new agent targeting Top2, and may suggest more effective use of Top2 targeting drugs that are in clinical use.